The present invention relates to the use of aminothiol compounds as linkers in preparing conjugate vaccines.
Covalent binding of a polysaccharide or other hapten to an immunogenic protein or peptide or other bio-organic molecule has proven to be a suitable method of preparing effective vaccines, for example against pathogenic organisms such as Haemophilus influenzae type b (meningitis, otitis media), Bordetella pertussis (whooping cough), Clostridium tetani (tetanus), meningococci (Neisseria meningitidis, meningitis, otitis media) and pneumoccocci (Streptococcus pneumoniae, pneumonia, meningitis, otitis media). Such conjugate vaccines have been described e.g. in U.S. Pat. No. 4,762,713. According to this US patent, binding between the polysaccharide and the carrier protein is performed by reductive amination of aldehyde or hemiacetal functions of the poly-saccharides with amino groups in the protein. Another suitable method of covalently binding a polysaccharide to a proteinaceous material is by activating hydroxyl functions to produce a side chain containing a function that can be coupled to the protein. Thus, the polysaccharide can be activated and then coupled to a thiol-bearing group such as cysteamine, which can be coupled to an activated amino acid in the protein. The use of cysteamine for coupling oligosaccharides to proteins has been described by Verheul et al (Infect. Immun. 59 (1991) 843-851). This use comprises activation of the saccharide by converting a carboxylic group to an N-succinimidyl ester (NSu), according to the following scheme:
Psxe2x80x94COOH+XONSuxe2x86x92Psxe2x80x94COxe2x80x94ONSu+HOXxe2x80x83xe2x80x83(1)
Psxe2x80x94COxe2x80x94ONSu+H2Nxe2x80x94CH2xe2x80x94CH2xe2x80x94Sxe2x80x94Sxe2x80x94CH2xe2x80x94CH2xe2x80x94NH2xe2x86x92xe2x86x92Psxe2x80x94COxe2x80x94NHxe2x80x94CH2xe2x80x94CH2xe2x80x94Sxe2x80x94Sxe2x80x94CH2xe2x80x94CH2xe2x80x94NH2+HONSuxe2x80x83xe2x80x83(2)
Psxe2x80x94COxe2x80x94NHxe2x80x94CH2xe2x80x94CH2xe2x80x94Sxe2x80x94Sxe2x80x94CH2xe2x80x94CH2xe2x80x94NH2+DTT-rdxe2x86x92xe2x86x92Psxe2x80x94COxe2x80x94NHxe2x80x94CH2xe2x80x94CH2xe2x80x94SH+HSxe2x80x94CH2xe2x80x94CH2xe2x80x94NH2+DTT-oxxe2x80x83xe2x80x83(3)
Psxe2x80x94COxe2x80x94NHxe2x80x94CH2xe2x80x94CH2xe2x80x94SH+Brxe2x80x94CH2xe2x80x94COxe2x80x94NHxe2x80x94Prxe2x86x92xe2x86x92Psxe2x80x94COxe2x80x94NHxe2x80x94CH2xe2x80x94CH2xe2x80x94Sxe2x80x94CH2xe2x80x94COxe2x80x94NHxe2x80x94Pr+HBrxe2x80x83xe2x80x83(4)
wherein Ps represents a polysaccharide, Pr represents a protein or peptide, and DTT-rd represents dithiothreitol in its reduced (dithiol) form and DTI-ox in its oxidised (1,2-dithiane) form. This approach, however, requires the presence of carboxyl groups in the polysaccharide, while many biologically interesting polysaccharides do not contain a carboxyl group.
It was found that polysaccharides can be effectively bound to cysteamine-like linkers without the need of other functional being present than hydroxyl groups by cyanogen bromide activation according to the following scheme.
Psxe2x80x94OH+Brxe2x80x94CNxe2x86x92Psxe2x80x94Oxe2x80x94CN+HBrxe2x80x83xe2x80x83(5)
Psxe2x80x94Oxe2x80x94CN+H2Nxe2x80x94CH2xe2x80x94CH2xe2x80x94Sxe2x80x94Sxe2x80x94CH2xe2x80x94CH2xe2x80x94NH2xe2x86x92xe2x86x92Psxe2x80x94Oxe2x80x94C(xe2x95x90NH)xe2x80x94HNxe2x80x94CH2xe2x80x94CH2xe2x80x94Sxe2x80x94Sxe2x80x94CH2xe2x80x94CH2xe2x80x94NH2xe2x80x83xe2x80x83(6)
Reaction (5) is followed by side reactions including a reaction with a second hydroxyl group to produce a cyclic imidocarbonate which can also result in coupling with an amino group as in reaction (6).
Thus the invention relates to a method of coupling a polysaccharide to another biopolymer wherein the polysaccharide is activated with a cyananting agent such as cyanogen halide and the activated polysaccharide is reacted with and aminothiol linker having formula 1
H2Nxe2x80x94[(CH2)mxe2x80x94CHR1xe2x80x94CR2R3xe2x80x94A]qxe2x80x94CHR4xe2x80x94(CHR5)pxe2x80x94CHR6xe2x80x94Sxe2x80x94R7
wherein
A is a direct bond or a group having the formula
xe2x80x94{Zxe2x80x94(CH2)mxe2x80x94CHR1xe2x80x94CHR2R3}nZxe2x80x94,
m is an integer from 0 to 5;
n is an integer from 0 to 3;
p is an integer from 0 to 2;
q is the integer 0 or 1;
R1 is hydrogen or C1-C6 alkyl, wherein the C1-C6 alkyl is optionally substituted by amino, hydroxyl, carboxyl, C1-C4 alkoxycarbonyl, carbamoyl, mono- or di-C1-C4-alkylcarbamoyl or N-(xcex1-carboxyalkyl)carbamoyl-; or if m xe2x89xa00, R1 is hydroxyl, amino or peptidyl-amino;
R2 and R3 are independently hydrogen or C1-C4 alkyl, or together from an oxo group;
R4 is hydrogen, C1-C4 alkyl, carboxly, C1-C4 alkoxycarbonyl, carbamoly, mono-or di-C1-C4-alkylcarbamoyl or N-(xcex1-carboxyalkyl) carbamoyl;
R5 is hydrogen, methyl, hydroxy or C1-C7 acyloxy;
R6 is hydrogen or methyl;
R7 is hydrogen or thiol-protecting group or group having the formula
xe2x80x94Sxe2x80x94CHR6xe2x80x94(CHR5)pxe2x80x94CHR4xe2x80x94[Axe2x80x94CR2R3xe2x80x94CHR1xe2x80x94(CH2)m]qxe2x80x94NH2;
xe2x80x83and
Z is imino, methylimino, oxygen or sulphur;
to produce a thiolated polysaccharide having the formula 2
Psxe2x80x94Oxe2x80x94C(xe2x95x90NH)xe2x80x94NHxe2x80x94[(CH2)mxe2x80x94CHR1xe2x80x94CR2R3xe2x80x94A]qxe2x80x94CHR4xe2x80x94(CHR5)pxe2x80x94CHR6xe2x80x94Sxe2x80x94R7xe2x80x83xe2x80x832
wherein Ps represents a polysaccharide residue and A, m, p, q, R1, R2, R3, R4, R5, R6 and R7 are as defined above, followed by optionally removing protecting group R7 and reacting the thiolated polysaccharide with an activated biopolymer. The preferred linkers are as defined in claims 3-8. A suitable example of the linkers is cysteamine or its oxidised form cystamine.
This method works satisfactorily for the majority of polysaccharides including most bacterial polysaccharides. However, no coupling to a useful degree is found with some polysaccharides such as the 19F type pneumococcal capsular polysaccharide.
Although the present inventors do not wish to be bound by any specific theory, one possible explanation for the incompleteness or failure of the cysteamine coupling is that the cysteamine adduct, once formed, may revert to the original materials by intramolecular displacement.
It has furthermore been found that any insufficient coupling can be solved by using amino-thiol linkers complying with formula 3,(=formula 1 with q=1)
H2Nxe2x80x94(CH2)mxe2x80x94CHR1xe2x80x94CR2R3xe2x80x94Axe2x80x94CHR4xe2x80x94(CHR5)pxe2x80x94CHR6xe2x80x94Sxe2x80x94R7
wherein
A is a direct bond or a group having the formula
xe2x80x94{Zxe2x80x94(CH2)mxe2x80x94CHR1xe2x80x94CHR2R3}nZxe2x80x94,
m is an integer from 0 to 5;
n is an integer from 0 to 3, preferably between 0 and 2;
p is an integer from 0 to 2;
q is the integer 0 or 1;
R1 is hydrogen or C1-C6 alkyl, wherein the C1-C6 alkyl is optionally substituted by amino, hydroxyl, carboxyl, C1-C4 alkoxycarbonyl, carbamoyl, mono- or di-C1-C4-alkylcarbamoyl or N-(xcex1-carboxyalkyl)carbamoyl; or, if mxe2x89xa00, R1 is hydroxyl, amino or peptidyl-amino;
R2 and R3 are independently hydrogen or C1-C4 alkyl, or together from an oxo group;
R4 is hydrogen, C1-C4 alkyl, carboxly, C1-C4 alkoxycarbonyl, carbamoly, mono-or di-C1-C4-alkylcarbamoyl or N-(xcex1-carboxyalkyl) carbamoyl;
R5 is hydrogen, methyl, hydroxy or C1-C7 acyloxy;
R6 is hydrogen or methyl;
R7 is hydrogen or thiol-protecting group or group having the formula
xe2x80x94Sxe2x80x94CHR6xe2x80x94(CHR5)pxe2x80x94CHR4xe2x80x94[Axe2x80x94CR2R3xe2x80x94CHR1xe2x80x94(CH2)m]qxe2x80x94NH2;
xe2x80x83and
Z is imino, methylimino, oxygen or sulphur, wherein R2 and R3 together can form an oxo group.
The amino-thiol linkers according to formula 3 may be straight or branched xcex1,xcfx89-aminothiol derivatives having at least 4 carbon atoms and optionally one or more heteroatoms in the chain, such as 4-aminobutanethiol, 5-aminopentanethiol, 2-(2-aminoethylaminoethanethiol and the like. Preferred compounds are those wherein H2Nxe2x80x94(CH2)mxe2x80x94CHR1xe2x80x94CR2R3xe2x80x94 represents an amino acid such as glycine, alanine, xcex2-alanine, serine, glutamine, xcex3-aminobutyric acid, lysine and xcex5-aminocaproic acid, or an oligopeptide such as Nxcex1-glycyl-lysine and higher homologues of Nxcex1-peptidyl-xcex1,xcfx89-diaminoacids. The group H2Nxe2x80x94(CH2)mxe2x80x94CHR1xe2x80x94CR2R3xe2x80x94Axe2x80x94 may also represent a linear oligopeptide such as glycylglycine.
In the linkers of both formula 1 and 3, the group Axe2x80x94CHR4xe2x80x94(CHR5)pxe2x80x94CHR6xe2x80x94Sxe2x80x94R7 may e.g. be derived from 2-aminoethanethiol (cysteamine), 2-mercaptoethanol, 1,2-ethanedithiol, 2-amino-2-methylpropanethiol, 3-aminopropanethiol, 2-hydroxy-3-aminopropanethiol, monothio- and dithio-threitol or -erythritol, cysteine, homocysteine and their esters or amides, and the like. The most preferred compounds according to formula 3 are N-glycyl-cysteamine and its disulphide precursor N,Nxe2x80x2-diglycyl-cystamine.
Most of the compounds complying with formula 1 and 3, such as N,Nxe2x80x2-di-glycylcystamine and N-alanyl-S-acetylcysteamine, are known from WO 85/00167 as radioprotective agents.
Biopolymers that can be conjugated by the present process comprise any macro-molecular (MW greater than 1 kDa) natural or nature-like compound containing hydroxyl, amino and/or mercapto groups. In particular, such biopolymers include natural or modified polysaccharides, natural, modified or synthetic peptides and proteins, lipoproteins, glycoproteins and nucleic acids. Most preferably the present linkers are used for conjugating one or more polysaccharides to a protein or peptide.
Polysaccharides that can be conjugated include starch-like and cellulosic material, but the present method is especially suitable for conjugating microbial poly-saccharides that are haptens or immunogens. Examples thereof are pneumococcal capsular polysaccharides of the various types including e.g. Danish types 1, 3, 4, 6A, 6B, 7S, 9V, 14, 18C, 19F and 23F, group B streptococcal polysaccharides, capsular polysaccharides of Klebsiella pneumoniae, Haemophilus influenzae including type b polysaccharide, Neisseria meningitidis (groups A and C), Pseudomonas aeruginosa or Escherichia coli. It is noted that the term xe2x80x9cpolysaccharidesxe2x80x9d as used herein comprises sugar-containing polymers and oligomers, whether they only contain glycosidic linkages or also phosphodiester or other linkages. They may also contain non-sugar moieties such as acid groups, phosphate groups, amino groups, sugar alcohols and amino acids, and they may be depolymerised or not. By way of illustration the repeating units of the pneumococcal capsular polysaccharides types 6B, 14, 19F and 23 and the H. influenzae type b capsular polysaccharide are given below:
Pn 6B xe2x86x922)-xcex1-D-Galp-(1xe2x86x923)-xcex1-D-Glcp-(1xe2x86x923)-xcex1-L-Rhap-(1xe2x86x924)-D-Ribitol -5-(PO4xe2x88x92xe2x86x92
Pn 14 xe2x86x924)-xcex2-D-Glcp-(1xe2x86x926)-xcex2-D-Glcp*NAc-(1xe2x86x923)-xcex2-D-Galp-(1xe2x86x92
*: bearing a xcex2-D-Galp-(1xe2x86x924) side group
Pn 19F xe2x86x924)-xcex2-ManpNAc-(1xe2x86x924)-xcex1-D-Glcp-(1xe2x86x922)-xcex1-L-Rhap-(1-PO4xe2x88x92xe2x86x92
Pn 23F xe2x86x924)-xcex2-D-Glcp#-(1xe2x86x924)-xcex2-D-Galpand-(1xe2x86x924)-xcex2-L-Rhap-(1xe2x86x92
#: bearing a phosphoglyceryl-(xe2x86x923) side group
and: bearing an xcex1-L-Rhap-(1xe2x86x922) side group
Hi b xe2x86x923)-xcex2-D-Ribf-(1xe2x86x921)-D-Ribitol-5-(PO4xe2x88x92xe2x86x92
A review of bacterial polysaccharides of interest can be found in: Lennart Kenne and Bengt Lindberg, xe2x80x9cBacterial polysaccharidesxe2x80x9d in The polysaccharides, Vol. 2, Ed. G. O. Aspinall, 1983, Ac. Press, pp. 287-363.
Proteins and peptides that may be conjugated with the present method include immunogenic and non-immunogenic proteins. Examples are serum albumins and various bacterial toxins and toxoids, such as diphtheria toxin, tetanus toxoid, pneumolysin, pneumolysoid, toxins of other organisms such as Pseudomonas, Staphylococcus, Bordetella pertussis, Escherichia coli, optionally detoxified, so-called cross-reacting material (e.g. CRM 197) and haemocyanins. They may also be outer membrane proteins of organisms such as Neisseria meningitidis or Bordetella pertussis. The proteins may also be antibodies to be used for conveying another biomaterial to a desired site. The proteins and peptides may be used as independent immunogens, or they may be used to render the other material such as haptens more immunogenic. They may native or detoxified or mutated. The term peptides and proteins are used indiscriminately herein, even though proteins in general denote higher molecular weight materials than peptides.
The linkers of formula 3 can be prepared by methods known per se, such as those described in EP-A-131500. For example, the linker compound can be prepared from a suitable amino-thiol, dithiol or mercapto-alcohol, according to the nature of group A, such as 2-aminoethanethiol (cysteamine), 3-aminopropanethiol or cysteine, wherein the thiol group is preferably protected e.g. by an acyl group or as a disulphide. If the group A contains a chain with the formula xe2x80x94(Zxe2x80x94CHR1xe2x80x94CHR2R3)nxe2x80x94, this can be introduced by reaction with e.g. ethyleneimine, propylene oxide, under the appropriate conditions to obtain an adduct having the desired value of n. Alternatively, if 13 (Zxe2x80x94CHR1xe2x80x94CHR2R3)nxe2x80x94 represents an oligopeptide chain, this group can be introduced by conventional peptide synthesis methods. The terminal group H2Nxe2x80x94(CH2)mxe2x80x94CHR1xe2x80x94CR2R3xe2x80x94 can be introduced by reaction of the appropriate-activated compound with the precursor HAxe2x80x94CHR4xe2x80x94(CHR5)pxe2x80x94CHR6xe2x80x94Sxe2x80x94R7. Where H2Nxe2x80x94(CH2)mxe2x80x94CHR1xe2x80x94CR2R3xe2x80x94 represents an amino acid residue such as glycine, alanine or lysine, it can be coupled again by conventional methods.
The use of the amino-thiol linkers comprises coupling of the linker, preferably with a protecting group on the thiol function, to a first biopolymer, especially a polysaccharide, which is optionally activated. Activation can be performed using known methods, such activation of a carboxyl groups on the polysaccharide (if present) e.g. with a carbodiimide, or by introduction of an aldehyde group, e.g. by oxidation. Advantageously, coupling is performed by reaction with cyanogen bromide. This results in the presence of reactive isocyanate groups, which readily react with the amino function of the linker to produce an isoureum bond. The CNBr activation is performed in such a manner that at least 0.1 activated site is introduced per repeating unit (RU) (in a polysaccharide: a repeating mono- or oligosaccharide unit). The coupling preferably yields 1 amino function per 1-10 RU.
The thiol-protecting group, if present, is then removed e.g. by reaction of the protecting disulphide with a reducing agent, for example a mercaptan such as 2-mercaptoethanol or dithiothreitol, or a trialkylphosphine. Reduction preferably results in 1 thiol group per 5-15 RU. The thiol function can then react with an active function of a second biopolymer, such as a bromoacyl group, an iodoacyl group, a pyridyldithio group or a maleimido-alkyl (or -aryl or -cycloalkyl) group, preferably bound to a lysine residue of the protein. The activated function can be introduced by chemical post-modification of the protein, such as reaction with N-succinimidyl-bromoacetate or N-(xcfx89-maleimidoalkyloxy)succinimide, or by peptide synthesis using one or more amino acid precurors already containing the active function.
The intermediates and the final conjugate can be purified as necessary using methods known per se, such as chromatography (ion exchange, hydrophobic interaction or affinity), gel filtration, dialysis, membrane filtration, selective precipitation using ammonium sulphate or alcohol, and the like.
The conjugates can be incorporated in a vaccine formulation in a manner known in the field of vaccination, using appropriate adjuvants, diluents, stabilisers, buffers, etc. The vaccines can be used in protecting humans against pathogens or in protecting animals. Alternatively the conjugates of appropriate biopolymers can be used in human or veterinary therapy or as a diagnostic agent.
Another advantageous use of the method of the present invention concerns immobilisation of proteins and peptides on a polysaccharide such as dextrans agarose, sepharose for the purpose of purification of antigens, antibodies and other biologically relevant molecules e.g. by affinity chromatography, or for use in immunoassays and the like.